Process for the preparation of androstane-3,17-dione derivatives

ABSTRACT

A process for the preparation of androstane-3,17-dione compounds of the formula ##STR1## wherein X is 1,2-methylene or 1- or 2-methyl, comprises fermenting a sterol of the formula ##STR2## wherein X is as above and R 1  is the hydrocarbon residue of 8-10 carbon atoms, of a sterol, with a microorganism culture capable of effecting the side chain degradation of sterols.

BACKGROUND OF THE INVENTION

Numerous microorganisms, for example, of the genera Arthrobacter,Brevibacterium, Microbacterium, Protaminobacter, Bacillus, Norcardia, orStreptomyces, especially Mycobacterium, are capable of degradingzoosterols and phytosterols to carbon dioxide and water. During thisdegradation, 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dioneare formed as intermediates.

It is possible, by using inhibiting additives or mutated microorganismsto control degradation of the sterols to prevent further degradation ofthe thus-formed 4-androstene-3,17-dione or 1,4-androstadiene-3,17-dione.See DOS's (German Unexamined Laid-Open Application) 1,543,269 and1,593,327, and U.S. Pat. No. 3,684,657.

SUMMARY OF THE INVENTION

This invention relates to a process for the preparation of anandrostane-3,17-dione compound of Formula I ##STR3## wherein X is1,2-methylene or 1- or 2-methyl, comprising fermenting a sterol ofFormula II ##STR4## wherein X is as above and R₁ is a saturated orunsaturated hydrocarbon sterol side chain of 8-10 carbon atoms with amicroorganism culture capable of degrading the sterol side chain.

DETAILED DESCRIPTION

A sterol hydrocarbon residue R₁ of 8-10 carbon atoms is an unsaturatedor hydrogenated side chain of a naturally occurring zoosterol orphytosterol, e.g., cholesterol, stigmasterol, campesterol,brassicasterol, or the sitosterols.

Sterol compounds of Formula II are, for example, compounds which ofFormula II a ##STR5## wherein X is as above and R₂ is hydrogen, methylor ethyl.

Suitable starting materials for the process of this invention are, forexample, sterols wherein X is 1α-methyl, 1β-methyl, 1α,2α-methylene, or1β,2β-methylene. Examples of suitable starting compounds are:

1α-methyl-5α-cholestan-3-one,

1β-methyl-5α-cholestan-3-one,

1α,2α-methylene-5α-cholestan-3-one,

1α-methyl-5α-stigmastan-3-one,

1α,2α-methylene-5α-stigmasten-3-one, or the corresponding sitosterolderivatives.

Although different starting compounds are used and the reaction can beconducted in the absence of inhibitors, the process of the invention isaccomplished under the same fermentation conditions utilized inconventional microbiological side chain degradation reactions ofsterols.

The fermentation is conducted using microorganism cultures customarilyemployed for the side chain degradation of sterols. Suitable culturesare, for example, those of the genera Arthrobacter, Brevibacterium,Microbacterium, Protaminobacter, Streptomyces. Those of the genusMycobacterium are preferred.

Examples of suitable microorganisms are: Microbacterium lactum IAM-1640,Protaminobacter alboflavus IAM-1040, Bacillus roseus IAM-1257, Bacillussphaericus ATCC-7055, Norcardia gardneri IAM-105, Morcardia minimaIAM-374, Norcardia corallina IFO-3338, Streptomyces rubescens IAM-74 orespecially the microorganisms Mycobacterium avium IFO-3082,Mycobacterium phlei IFO-3158, Mycobacterium phlei (Institute of Health,Budapest No. 29), Mycobacterium phlei ATCC-354, Mycobacterium smegmatisIFO-3084, Mycobacterium smegmatis ATCC-20, Mycobacterium smegmatis(Institute of Health, Budapest No. 27), Mycobacterium smegmatisATCC-19979, Mycobacterium fortuitum CBS-49566, Mycobacterium spec.NRRL-B-3805, and Mycobacterium spec. NRRL-B-3683. Mycobacterium spec.NRRL-B-3805 is most preferred.

Submerged cultures are grown under conditions customarily employed forthese microorganisms, using a suitable nutrient medium with aeration.Then, the substrate, dissolved in a suitable solvent or preferably inemulsified form, is added to the culture and the fermentation isconducted until maximum substrate conversion has been attained.

Suitable solvents for the substrate are, for example, methanol, ethanol,glycol monomethyl ether, dimethylformamide, or dimethyl sulfoxide. Thesubstrate can be emulsified, for example, by adding micronized substrateor substrate dissolved in a water-miscible solvent, e.g., methanol,ethanol, acetone, glycol monomethyl ether, dimethylformamide, ordimethyl sulfoxide, through nozzles under strongly turbulent conditions,to, preferably decalcified, water containing the customary emulsifyingagents. Suitable emulsifying agents include nonionic emulsifiers, forexample, ethylene oxide adducts or fatty acid esters of polyglycols.Examples of suitable emulsifiers are surfactants commercially available,as "Tegin", "Tween", and "Span".

The optimum substrate concentration, time of substrate addition, andduration of fermentation depend on the structure of the substrateemployed and on the type of the microorganism utilized. These variablesmust be determined in each individual case by means of preliminaryexperiments well-known to those skilled in the art.

It is surprising to those skilled in the art that, under otherwiseconventional conditions, the side chains of sterols of Formula II aredegraded, because it is known that side chain degradation of sterols iseffected by a very complex fermentation system. It is unexpected thatthe enzymes taking part in the side chain degradation of naturalsteroids also cause side chain degradation of the sterol derivatives ofFormula II, which do not occur in nature. Moreover, it could not beforeseen that enzyme systems effecting degradation of1,4-androstadiene-3,17-dione and of 4-androstene-3,17-dione areincapable of further degrading androstane-3,17-dione derivatives ofFormula I.

Androstane-3,17-dione compounds of Formula I which can be produced bythe process of the present invention are valuable intermediates for thesynthesis of pharmacologically active steroids, e.g.,17β-hydroxy-1α-methyl-5α-androstan-3-one,17β-hydroxy-1-methyl-5α-androst-len-3-one,2α-methyl-17β-propionyloxy-5α-androstan-3-one and1,2α-methylene-17α-hydroxy-4,6-pregnadiene3,20-dione. The 17-keto groupof the androstane-3,17-dione compounds can be reduced optionally afterketalizing the 3-oxo group. Also, the 17-keto group can be reacted withan organometallic compound of the formula

    MeR.sub.4

wherein R₄ is alkyl, alkenyl or alkinyl of up to 4 carbon atoms and Meis an alkali metal atom or a magnesium halide residue. After cleavingthe ketal group, which may be present, 17β-hydroxyandrostan-3-onecompounds of Formula III ##STR6## wherein X is as above and R₃ is R₄ orhydrogen, are obtained.

When R₃ is alkyl, alkenyl or alkinyl, it is preferably methyl, ethyl,vinyl or ethynyl.

The 17-keto group of androstane-3,17-dione compoudns of Formula I isreduced by methods well-known to those skilled in the art. See, forexample, John Fried: Organic Reactions in Steroid Chemistry, vanNostrand Reinhold Comp., New York, etc. (1972) I : 61 et seq. Thesecompounds can be reacted, for example, after ketalization, with sodiumborohydride or lithium aluminum hydride, to obtain, after cleaving theketals, the corresponding 17β-hydroxyandrostan-3-one derivatives ofFormula III which possess an anabolic and/or androgenic activity.

Methods for alkylating the 17-keto group are likewise known. See, forexample, John Fried: Organic Reactions in Steroid Chemistry, vanNostrand Reinhold Comp., New York, etc. (1972) 2 : 53 et seq.Androstane-3,17-dione derivatives of Formula I can be reacted,optionally after ketalizing the 3-oxo group, with alkyl magnesiumhalides, vinyllithium, or alkali metal acetylides. After removing theoptionally present ketal group, the 17αR-17β-hydroxyandrostan-3-onederivatives of Formula III are obtained. These compounds arepharmacologically active substances or intermediates for the preparationof pharmacologically active steroids, e.g.,17β-hydroxy-17α-methyl-5α-androstan-3,2 c-pyrazole.

The sterols of Formula II utilized as starting compounds can be producedfrom the corresponding naturallyoccurring sterols by meansconventionally utilized for introducing substituents at the 1- and/or2-positions of steroids. References to these conversions are: John Friedand John A. Edwards: Organic Reactions in Steroid Chemistry; vanNostrand Reinhold Comp., New York, 1972.

The following references on fermentation processes describe techniquescustomary in the art: (G. S. Fonken and R. A. Johnson: ChemicalOxydations with Microorganism: Macel Dekker Inc., New York, 1972.

The following examples serve to explain the invention.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative and not limitative ofthe remainder of the disclosure in any way whatsoever.

(A) Examples Concerning the Microbiological Side Chain DegradationEXAMPLE 1

A 2-liter Erlenmeyer flask containing 500 ml. of a sterile nutrientmedium containing 1% yeast extract, 0.45% disodium hydrogen phosphate,0.34% potassium dihydrogen phosphate, and 0.2% "Tween" 80, adjusted topH 6.7, is inoculated with a suspension of a Mycobacterium spec.NRRL-B-3805 dry culture and shaken for three days at 30° C. at 190r.p.m.

Twenty Erlenmeyer flasks, each containing 100 ml. of a sterile nutrientmedium, containing 2.0% corn steep liquor, 0.3% diammonium hydrogenphosphate, and 0.25% "Tween" 80, adjusted to pH 6.5, are each inoculatedwith 5 ml. of the Mycobacterium spec. growth culture and shaken for 24hours at 30° C. with 220 r.p.m.

Then, each culture is combined with 50 mg. of1α-methyl-5α-cholestan-3-one dissolved in 1 ml. of dimethylformamide.The fermentation is continued for another 96 hours at 30° C.

The combined cultures are extracted with ethylene chloride. The extractis concentrated under vacuum. The residue is purified by chromatographyover a silica gel column, thus obtaining after recrystallization fromdiisopropyl ether 0.5 g. of 1α-methyl-5α-androstane-3,17-dione, m.p.140° C.

Preparation of the Starting Compound:

(a) 97 g. of 3β-hydroxy-5α-cholestane is made into a slurry with 1.5 1.of acetone, and under agitation, this slurry is combined within 20minutes with 125 ml. of chromosulfuric acid, prepared from 267 g. ofchromium (VI) oxide, 400 ml. of water, 230 ml. of concentrated sulfuricacid, and brought with water to 1000 ml. The mixture is then stirred for1 hour at room temperature and stirred into ice water. Thethus-precipitated product is filtered off. After drying, the product isrecrystallized from diisopropyl ether, thus obtaining 72 g. of5α-cholestan-3-one, m.p. 127.5°-128.5° C.

(b) 50 g. of 5α-cholestan-3-one is dissolved in 750 ml. of acetic acidand 5000 ml. of ether, combined with 1 ml. of hydrobromic acid in aceticacid (37% strength), and gradually added dropwise to a solution of 22 g.of bromine in 50 ml. of acetic acid. The mixture is agitated for another30 minutes, diluted with methylene chloride, and washed in successionwith water, sodium bicarbonate solution, and water. After drying andevaporation, 61.5 g. of crude 2α-bromo-5α-cholestan-3-one is obtained.

(c) 61.5 g. of crude 2α-bromo-5α-cholestan-3-one is agitated in 615 ml.of dimethylformamide with 29.3 g. of lithium carbonate and 34.5 g. oflithium bromide for 20 hours at 100° C. The mixture is then stirred intoice water. The thus-precipitated product is filtered off, washedthoroughly with water, and taken up in methylene chloride. The residueobtained after drying and evaporation is chromatographed on silica gel.Recrystallization from methanol yields 32 g. of 5α-cholest-1-en-3-one,m.p. 99.5°-100° C.

(d) 6.5 g. of magnesium filings are reacted in 160 ml. of absolute etherwith 22 ml. of methyl iodide in 40 ml. of absolute ether to obtainmethylmagnesium iodide. Under ice cooling, 405 ml. of absolutetetrahydrofuran is added thereto, and the ether is distilled off up to aboiling point of 62° C. Under agitation under nitrogen, 1.3 g. of copper(I) chloride is added to the Grignard solution, which had been cooled inan ice bath. 25 g. of 5α-cholest-1-en-3-one dissolved in 81 ml. ofabsolute tetrahydrofuran is added dropwise thereto. The mixture is thenstirred for another 30 minutes with cooling, and the excess reagent isdecomposed with saturated ammonium chloride solution. Thereafter, themixture is diluted with ether. The aqueous phase is separated, and theether phase is washed with saturated ammonium chloride solution andwater. After drying and evaporation, the residue is chromatographed onsilica gel and, after recrystallization from methanol, 16.5 g. of1α-methyl-5α-cholestan-3-one is thus obtained, m.p. 132.5°-133° C.

EXAMPLE 2

Under the conditions of Example 1, 50 mg. portions of1α,2α-methylene-5α-cholestan-3-one (German Pat. No. 1,183,500) arereacted in twenty Erlenmeyer flasks with a Mycobacterium spec.NRRL-B-3805 culture and then worked up, thus obtaining1α,2α-methylene-5α-androstane-3,17-dione.

(B) Examples Concerning the Chemical Further Processing of theAndrostane-3,17-dione Derivatives EXAMPLE 1

7.2 g. of 1α-methyl-5α-androstane-3,17-dione dissolved in 150 ml. ofabsolute methanol is combined with 100 mg. of p-toluenesulfonic acid andrefluxed for 20 minutes. The solution is cooled and combined with 5 ml.of 10% sodium hydroxide solution and thereafter reduced at 0° C. byadding 1 g. of finely pulverized sodium borohydride in incrementalportions. After this addition (20 minutes), the mixture is agitated for2 hours with ice cooling and then combined with 50 ml. of 1N sulfuricacid and refluxed for one hour. The suspension is then stirred into 300ml. of ice-water-sodium chloride. The thus-precipitated product isvacuum-filtered, dried at 50° C. under vacuum, and recrystallized fromdiisopropyl ether, thus obtaining 4.15 g. of17β-hydroxy-1α-methyl-5α-androstan-3-one, m.p. 203°-205° C., which has apronounced androgenic activity (German Pat. No. 1,152,100).

EXAMPLE 2

Under the conditions of Example 1, 2.8 g. of1α,2α-methylene-5α-androstane-3,17-dione is reduced with sodiumborohydride. After the reaction mixture has been worked up, the yield is1.3 g. of 17β-hydroxy-1α,2α-methylene-5α-androstan-3-one (German Pat.No. 1,183,500).

The preceding examples can be repeated with similar success bysubstituting the generically and specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A process for the preparation of anandrostane-3,17-dione compound of the formula ##STR7## wherein X is1,2-methylene or 1- or 2-methyl, comprising fermenting; in the absenceof 4 androstene-3,17-dione degradation inhibitors, a sterol of theformula ##STR8## wherein X is as above and R₁ is a saturated orunsaturated hydrocarbon sterol side chain of 8-10 carbon atoms with amicro-organism culture capable of degrading the sterol side chain. 2.The process of claim 1, comprising the further step of either (a)selectively reducing the 17-keto group of the thus-producedandrostane-3,17-dione or (b) selectively reacting the 17-keto group ofthe thus-produced androstane-3,17-dione with an organometallic compoundof the formula MeR₄ wherein R₄ is alkyl, alkenyl or alkynyl of up to 4carbon atoms and Me is an alkali metal atom or a magnesium halideresidue, to produce a 17β-hydroxyandrostan-3-one compound of the formula##STR9## wherein X is 1,2-methylene or 1- or 2-methyl and R₃ is hydrogenor R₄, respectively.
 3. The process of claim 2, comprising theadditional steps of ketalizing the carbonyl at the 3-position of theandrostane-3,17-dione compound prior to the selective reduction orselective reaction and subsequently cleaving the ketal at the 3-positionof the 17β-hydroxyandrostan-3-one compound after the selective reductionor selective reaction.
 4. The process of claim 2, wherein R₃ ishydrogen, methyl, ethyl, vinyl or ethinyl.
 5. The process of claim 1,wherein the microorganism culture is of the genera Arthrobacter,Brevibacterium, Microbacterium, Protaminobacter, Bacillus, Norcardia orStreptomyces.
 6. The process of claim 1, wherein the microorganismculture is of the genus Mycobacterium.
 7. The process of claim 1,wherein the microorganism culture is Mycobacterium spec. NRRL-B-3805. 8.The process of claim 2, wherein the microorganism culture is of thegenera Arthrobacter, Brevibacterium, Microbacterium, Protaminobacter,Bacillus, Norcarida or Streptomyces.
 9. The process of claim 2, whereinthe microorganism culture is of the genus Mycobacterium.
 10. The processof claim 2, wherein the microorganism culture is Mycobacterium spec.NRRL-B-3805.